Method for obtaining plants exhibiting enhanced resistance to water stress

ABSTRACT

A method for obtaining a plant having a modified amount of ASR protein, providing it with enhanced resistance to water stress compared to a non-transformed plant, comprises: transforming at least a plant cell with a vector, containing an expression cassette including a nucleotide sequence coding for an ASR protein or inhibiting the expression of an ASR protein; culturing the resulting transformed cell so as to generate a plant containing in its genome the expression cassette.

[0001] The present invention relates to a method for obtaining plantsexhibiting enhanced resistance to water stress.

[0002] In temperate regions, periods of low rainfall, which vary inintensity and are unpredictable, are the cause of a notable decrease inproductivity in crop plants. The water deficit can severely affect plantgrowth and reproduction. Various strategies making use of physiologicalmechanisms (decrease in growth of aerial parts, closure of stomata)and/or cellular mechanisms (osmotic adjustment) may enable them to evadethis water stress, or at the very least to tolerate it. Thesemechanisms, which are at least partly controlled by ABA (abscisic acid),a phytohormone the concentration of which increases in plants subjectedto water stress (Zeevaart and Creelman, 1988), involve many proteinswith different putative functions: proteins of the membrane channeltype, or proteins expressed in response to damage caused to the cell(proteases, protease inhibitors), or else proteins whose functions arenot directly related to stress but which are expressed at higher levelsunder conditions of water or salinity stress (enzymes of glycolysis, ofmethionine synthesis). The plant's response depends, moreover, onenvironmental (type of restraint, intensity, duration) and genetic(species and genotype) parameters, making it difficult to determine therole of these proteins in the mechanisms of tolerance to drought.

[0003] There is therefore a real need to demonstrate the function ofcandidate genes in the mechanisms of tolerance to water stress, in orderto use them in transgenesis aimed at obtaining plants exhibiting bettertolerance to water stress. This is particularly important for field cropspecies, such as maize, for example, which attains a level of maximumsensitivity to drought 15 days before and 15 days after flowering of theplant (July-August in Europe), a period during which the plant wouldabsorb 45% of its total needs.

[0004] A first study carried out by Riccardi et al. (1998) on a maizeline designated Io (Iodent) has demonstrated about twenty proteins, theexpression of which is significantly increased in response to waterstress, and for which putative functions have been proposed.

[0005] One of these proteins, demonstrated in the Io line used byRiccardi et al. (1998) but not in the F2 line, exhibits homologies witha tomato protein which is induced by ABA, water stress and ripening ofthe fruit, ASR1 (ABA-water stress-ripening-induced protein), but thefunction of which is still unknown (Iusem et al., 1993). Other genesshowing homologies with tomato ASR have been identified, in particularin potato (Silhavy et al., 1995), lemon (Canel et al., 1995) and rice(Thomas et al., 1999), but no function has been clearly demonstrated forthe proteins encoded by these genes. A QTL (Quantitative Trait loci)study has made it possible to map the Asr1 gene in a region containingboth the locus controlling the amount of ASR1 and QTLs for foliarsenescence and protandry (De Vienne et al., 1999).

[0006] The authors of the present invention have now demonstrated therole of a recombinant ASR protein in the direct response of a plant towater stress, in order to use transgenesis. Specifically, they havesucceeded in developing a method for obtaining a plant having a modifiedamount of ASR protein, conferring on it better resistance to waterstress compared to a nontransformed plant.

[0007] A subject of the invention is therefore a method for obtaining aplant containing a modified amount of ASR protein, conferring on itbetter resistance to water stress compared to a nontransformed plant,comprising the steps consisting in:

[0008] transforming at least one plant cell with a vector containing anexpression cassette comprising a nucleotide sequence encoding an ASRprotein or blocking the expression of an ASR protein;

[0009] culturing the cell thus transformed so as to generate a plantcontaining, in its genome, said expression cassette.

[0010] The term “ASR protein” is intended to mean a protein expressednaturally by a plant in response to water stress, having an amino acidsequence identical or homologous to the maize sequence SEQ ID No 2.

[0011] The term “homologous sequence” is intended to mean preferably asequence exhibiting at least 50%, preferably 70%, similarity with thesequence SEQ ID No 2.

[0012] For the purpose of the invention, included in the definition of“ASR protein” are all the ASR proteins of various plants, such as, forexample, that of rice, and also the proteins which are modified, forexample by addition, deletion and/or substitution (preferablyconservative substitution) of a small number of amino acids.

[0013] Also included in the definition of “ASR protein” are thepolypeptides encoded by all or part of the sequence SEQ ID No 1, No 3,No 4 or No 5, or any homologous sequence, it being understood that thesepolypeptides conserve the property of resistance to water stress.

[0014] The nucleic acid sequence encoding an ASR protein may moreparticularly be a sequence from a cereal, in particular a sequence frommaize.

[0015] Said sequence may advantageously be a cDNA sequence specific forthe state of water stress, isolated from a maize line by differentialhybridization. Such a sequence is given in the attached sequencelisting, and designated SEQ ID No 1. It is also possible to use agenomic DNA sequence encoding a maize ASR protein, as defined by one ofthe sequences SEQ ID No 3, SEQ ID No 4 and SEQ ID No 5, or a sequencehomologous thereto, provided that it is expressed in modified amountscompared to the amount usually produced by a nontransformed plant.

[0016] In the attached sequence listing,

[0017] SEQ ID No 3 is a genomic DNA sequence isolated from a maize linewhich strongly expresses the ASR protein,

[0018] SEQ ID No 4 is a genomic DNA sequence isolated from a controlmaize line A188, and

[0019] SEQ ID No 5 is a genomic DNA sequence isolated from an F₂ maizeline.

[0020] Said sequence may in particular encode the amino acid sequenceSEQ ID No 2 of the maize ASR protein, or a variant thereof, for examplea variant having the sequence SEQ ID No 2 with an insertion of a lysineresidue between amino acids 55 and 56 of SEQ ID No 2.

[0021] It is also possible to use nucleotide sequences encoding ASRsfrom other plants, such as, for example, those cited above.

[0022] The nucleic acid encoding an ASR protein is inserted into anucleic acid construct, called expression cassette, and is functionallylinked to elements which allow the expression thereof and, optionally,the regulation thereof.

[0023] Among these elements, mention may be made of promoters,activators and terminators of transcription.

[0024] Use may preferentially be made of a constitutive promoter, suchas the rice actin promoter, followed by the rice actin intron (RAP-RAI)contained in the plasmid pAct1-F4 (Mc Elroy et al., 1991) or the ³⁵Spromoter (Kay et al., 1987), or a tissue-specific promoter. By way ofexample, mention may be made of the wheat HMWG promoter or the radishcruciferin gene promoter, PCRU, which both allow expression of theprotein of interest in the seeds (Anderson O. D. et al., 1989;Depigny-This et al., 1992). Use may advantageously be made of promotersequences which induce expression under water conditions (Kasuga et al.,1999). Among the terminators which can be used in the constructs of theinvention, mention may in particular be made of the 3′ end of theAgrobacterium tumefaciens nopaline synthase gene (Depicker et al.,1982). Mention may also be made of the ³⁵S polyA terminator of thecauliflower mosaic virus (CaMV), described in the article by Franck etal. (1980).

[0025] The expression of the ASR protein can also be regulated by usingsequences such as peptide addressing signals (chloroplast addressingsignals, vacuolar addressing signals, addressing signals for endoplasmicretention, etc.), or such as intron sequences, enhancer sequences orleader sequences.

[0026] In the present invention, the sequence of interest may be anucleotide sequence encoding an ASR protein, said sequence being placedin the sense direction, or a nucleotide sequence blocking the expressionof an ASR protein. This nucleotide sequence blocking the expression ofthe ASR protein is preferentially a sequence encoding all or part of theASR protein, said sequence being placed in the antisense direction. Whenthe sequence encoding an ASR protein is placed in the sense direction, atransformed plant is obtained which exhibits an increase in the ASRprotein compared to a nontransformed plant. This plant has the advantageof withstanding water stress more effectively than a nontransformedplant. When the sequence is placed in the antisense direction, atransformed plant is obtained which also exhibits a modification ofresistance to water stress. Without adhering in any way to a precisemechanism of action, this observation might be explained by the plantactivating an alternative pathway of resistance to water stress, inresponse to inhibition of the ASR by the antisense sequences.

[0027] The expression cassette is inserted into a nucleotide vector,such as a plasmid, which may also comprise a marker gene, for example agene making it possible to select between a transformed plant and aplant which does not contain the transfected foreign DNA. As markergene, mention may be made of a gene which confers resistance to anantibiotic, for example to hygromycin (Herrera-Estrella et al., 1983) orresistance to a herbicide such as the sulfonamide asulam (WO 98/49316).

[0028] This vector or any sequence encoding an ASR protein, such as thesequences SEQ ID No 1, SEQ ID No 3, SEQ ID No 4 and SEQ ID No 5, orsequences homologous to the latter, can be used to transform plant cellsaccording to techniques commonly known to those skilled in the art, inorder to obtain plants exhibiting enhanced resistance to water stress.

[0029] According to one embodiment of the method of the invention, theplant cells are transformed with a vector as defined above, transferredinto a cellular host capable of infecting said plant cells by allowingintegration into the genome of the latter of the nucleotide sequences ofinterest initially contained in the genome of the abovementioned vector.Advantageously, the cellular host used is a bacterial strain, such asAgrobacterium tumefaciens, in particular according to the methoddescribed in the article by An et al. (1986), or else Agrobacteriumrhizogenes, in particular according to the method described in thearticle by Guerche et al. (1987).

[0030] For example, the plant cells can be transformed by transferringthe T region of the Agrobacterium tumefaciens extrachromasomal,circular, tumor-indicating Ti plasmid, using a binary system (Watson etal., 1994). To do this, two vectors are constructed. In one of thesevectors, the T region has been removed by deletion, with the exceptionof the left and right borders, a marker gene being inserted between themso as to allow selection in the plant cells. The other partner of thebinary system is a helper Ti plasmid, which is a modified plasmid whichno longer has a T region but which still contains the vir virulencegenes required for transformation of the plant cell.

[0031] According to a preferred mode, use may be made of the methoddescribed by Ishida et al. (1996), for the transformation ofmonocotyledons.

[0032] Other embodiments of the method of the invention may also bementioned, in particular methods of direct gene transfer into plantcells, such as direct microinjection into plant embryoids (Neuhaus etal., 1987), infiltration under vacuum (Bechtold et al., 1993) orelectroporation (Chupeau et al., 1989), or else direct precipitationusing PEG (Schocher et al., 1986) or bombardment with particles coveredwith the plasmid DNA of interest, using a particle gun (M. Fromm et al.,1990).

[0033] According to another protocol, the transformation is carried outaccording to the method described by Finer et al. (1992), using atungsten or gold particle gun.

[0034] The subject of the invention is also a host cell transformed withthe nucleic acid sequences described above, and also a plant or part ofa plant, in particular fruit, seed, grain, pollen, leaf or tuber, whichcan be obtained using one of the methods set out above.

[0035] They may, for example, be field crop plants (wheat, rapeseed,sunflower, peas, soybean, barley, in particular maize, etc.) orvegetables and flowers.

[0036] The hybrid transgenic plants, obtained by crossing at least oneplant according to the invention with another, are also part of theinvention.

[0037] The invention in particular relates to a plant exhibiting anincrease in expression of the ASR protein compared to a nontransformedplant, for example a 2- to 3-fold increase. This increase in expressionof the ASR protein confers enhanced resistance to water stress on thetransformed plants.

[0038] The resistance to water stress of the transformed plantsaccording to the invention, compared to the control plants, can beassessed using various morphological, physiological and/or biochemicalmeasuring methods, for particular irrigation conditions. By way ofexample, the tolerance to stress can be measured by phenotypicobservation (i) of foliar senescence, by morphological measurements andby assaying the chlorophyll in the foliar disks, (ii) of the protandryor date of flowering of the male and female plants, (iii) of the growthof the plant, by measuring the final length and width of the leaves andalso the final height of the plant, and by studying the rolling up ofthe leaves, or else (iv) of the yield of grain, of the weight of athousand grains and of the number of ears per plant.

[0039] The stress experienced by the plants can also be evaluated bymeasuring the ABA content (method of Quarrie et al., 1988) or bymeasuring the water potential, or else, where appropriate, by monitoringexpression of the protein by two-dimensional electrophoresis using aleaf sample.

[0040] The plants obtained according to the invention can also be usedin allele complementation experiments in order to validate the functionof the inserted gene. Use of the transformants in backcross experimentsmakes it possible to introduce only the gene inserted in the parentalgenetic background, without other sequences which might influence thephenotype of the recombinant with regard to tolerance to drought.

[0041] Preferably, the inserted gene is coupled with a selectable markergene, which facilitates the monitoring of the backcrosses and,consequently, the monitoring of the insertion of the gene of interestinto the line in which it is desired to validate the effect.

[0042] The principle consists in crossing the transformant with theparental line not possessing the favorable allele of the gene ofinterest, and comparing the phenotypes of the recombinant line with theparental lines. It is also possible to use transformants containing thegenomic sequences in these complementation experiments. Thiscomplementation assay makes it possible to verify in particular thatoverexpression of the cDNA in the sense direction complements the effectof the weak (or null) allele.

[0043] Thus, it is, for example, possible to verify that the ASR1 geneis the gene responsible for the QTL and PQL (protein quantitative loci),found at this genetic position on chromosome 10 in maize. Since theamount of this protein varies between different lines, strongerexpressions; or even expressions of more favorable alleles, can bedetected and exploited for improving plants. Plants exhibiting favorablealleles can be detected by immunoassays with antibodies specific for theASR1 protein (ELISA assay, Western blotting, etc.).

[0044] The use of a nucleic acid encoding an ASR, of a fragment of thisnucleic acid, as a probe or primer for PCR-type amplification, in orderto select transformed plants exhibiting better resistance to waterstress, also falls within the context of the invention.

[0045] The nucleic acid sequences encoding an ASR, such as thosedesignated SEQ ID No 1, SEQ ID No 3, SEQ ID No 4 and SEQ ID No 5, andalso any oligonucleotide obtained from one of these sequences, can thusbe used as probes in marker-assisted selection programs, for example forfollowing the introgression of the gene encoding the maize ASR proteininto a plant. For this, at least one of these probes is labeled, forexample with a radioactive isotope, and then brought into contact withgenomic DNA from the plant, predigested with restriction enzymes, underconditions which allow specific hydration of the labeled probe to theDNA in question. Other techniques using for example PCR may also be usedto carry out genotyping.

[0046] The following figures and examples illustrate the inventionwithout limiting the scope thereof.

LEGEND TO THE FIGURES

[0047]FIG. 1 represents a restriction map of the plasmid pWP 280containing the promoter pActin intron-barnase-Nos PolyA.

[0048]FIG. 2 represents a restriction map of the intermediate vectorpBIOS 308 containing ZmASR1 cDNA in the sense direction.

[0049]FIG. 3 represents a restriction map of the intermediate vectorPBIOS 309 containing ZMASR1 cDNA in the antisense direction.

[0050]FIG. 4 represents the effect of each “antisense” or “sense”transformation event, relative to its own control (Basta sensitive), onfoliar senescence, the effect being measured on the first day of gradingafter flowering.

[0051]FIG. 5 represents kinetics of the effect, for all the “sense”,“nontransformed” and “antisense” events, possibly placed underconditions of water stress, on foliar senescence.

EXAMPLES Example 1

[0052] Obtaining and Cloning the cDNA of ZmAsr1 from a Maize Line WhichStrongly Expresses the ASR Protein

[0053] a) Culture Conditions and Taking of Samples for the Plants HavingBeen Used to Isolate the cDNA

[0054] The authors of the invention cloned the ZmAsr1 cDNA from Io maizelines (Riccardi et al., 1998) or from maize lines selected according tothe same criteria as Io.

[0055] The maize plants are grown in perlite under controlled conditionsin a culturing chamber (illumination: 450 mmol m⁻² s⁻¹, photoperiod: 16h, day/night temperature: 25° C./20° C., relative humidity 60%), wateredwith a nutrient solution. When the plants have reached the “5 leaf”stage (5th emerged leaf), the watering is either stopped, for the plantsunder conditions of water stress, or it is continued, for the controlplants. Ten days later, samples are taken from the ensheathed part ofthe blade of the 7th leaf.

[0056] b) Isolating the cDNAs Specific for the State of Water Stress byDifferential Hybridization

[0057] A cDNA library is prepared from mRNA from the leaves of stressedplants using the Lambda ZapII cDNA synthesis/Gigapack GoldI cloning kit(Stratagene, La Jolla, USA), according to the manufacturer'sintructions.

[0058] The mRNAs prepared from the leaves of stressed plants and ofcontrol plants are transcribed into radiolabeled cDNA using 100 μCi of³²P-dATP and hexanucleotides serving as random primers (Sambrook et al.,1989). The single-stranded cDNAs, originating either from the stressedplant or from the control plant, are hybridized, in the same proportion,on the cDNA clones of the library. The hybridization temperature in thephosphate buffer/SDS/EDTA system (Church and Gilbert, 1984) is 68° C.and the final washes are carried out with solutions containing 0.1×SSC,0.05% (weight/volume) SDS.

[0059] The labeled clones are recovered by in vivo excision of thephagemid according to the Stratagene protocol using E. coli SOLR and the“Exassist” helper phage. The DNA of these clones is sequenced andcompared to nucleic acid sequence libraries (BLAST) according to themethod described in Altschul et al. (1990). One of these clones exhibitsstrong homology with tomato Asr1 and is named ZmAsr1 for Zea maize Asr1.

Example 2

[0060] ZmASR1 Genomic Sequences

[0061] The primers cASR1-1F (5′-TGTCGATCCAATTGTCACTT-3′)=SEQ ID No 6 andcASR1-740R (5′-TGGAGAAACGTAAACAACTA-3′)=SEQ ID No 7, defined at the twoends of the cDNA sequence of the ASR1 protein, are used in PCRamplification on maize line total DNA. The PCR reactions are carried outaccording to conventional techniques.

[0062] The PCR products are analyzed by electrophoresis: a band at 900bp is recovered in order to extract the DNA therefrom and clone itaccording to conventional techniques. After verification of their size,the inserts are extracted and sequenced.

Example 3

[0063] Construction of Chimeric Genes for the Constitutive Expression ofthe ASR1 Protein or Else of an Antisense Leading to Inhibition Thereof.

[0064] First, 2 basic plasmid vectors are constructed, pBIOS 306 andpBIOS 307, containing the actin promoter-actin intron (pAct), the cDNAof the Asr1 gene, respectively in the sense and antisense direction, andthe nopaline synthase terminator (terNos) which introduces apolyadenylation signal which is functional in many plant species.

[0065] Intermediate vectors are then produced for homologousrecombination with the Japan Tobacco vector pSB1 (EP 672 752) inAgrobacterium tumefaciens strain LBA 4404 (Hoekema et al., 1983).

[0066] The transfer followed by the expression of the genes (gene forselection and gene of interest) into maize is based on the naturalproperties of Agrobacterium tumefaciens (Zambrisky et al., 1989) and onthe superbinary plasmid strategy (Hiei et al., 1994 and Ishida et al.,1996).

[0067] The restriction enzymes used for the cloning are provided by NewEngland Biolabs (New England Biolabs, UK). The enzymatic reactions arecarried out by following the protocols described, by Sambrook et al., inthe molecular cloning manual (Molecular Cloning: A Laboratory Manual,1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0068] a) Construction of the Basic Plasmid Vectors for the ConstitutiveExpression of the Asr1 Gene and of its Antisense

[0069] The molecular constructs for the constitutive expression of theAsr1 gene in the sense and antisense direction were prepared asdescribed below:

[0070] cloning of the 795 bp I/XhoI fragment (Asr1 cDNA=SEQ ID No 1)into the EcoRV-restricted vector pBIOS 298.

[0071] The vector pBIOS 298 contains the actin promoter-actin intron(pAct) (Mc Elroy et al., 1991) and the Nos terminator. This vector wasgenerated by deletion of the 366 bp PstI fragment (Barstar gene) of thevector pWP 280, containing the pActin-intron˜Barstar˜Nos polyA cassette(FIG. 1).

[0072] This nonoriented cloning makes it possible to obtain 2 newvectors:

[0073] the vector pBIOS 306 carrying the gene pAct-ZmAsr1 sense-terNos

[0074] the vector pBIOS 307 carrying the gene pAct-ZmAsr1antisense-terNos.

[0075] The orientation of the cDNA relative to the actin promoter wasdetermined by simple enzymatic restriction with EagI and doubleenzymatic restriction with Hind III-EcoRV.

[0076] b) Construction of the Intermediate Vectors for HomologousRecombination with pSB1 (Obtaining Superbinary Plasmids)

[0077] The vectors used for the homologous recombination inAgrobacterium tumefaciens are derived from the vector pBIOS 273.

[0078] Construction of the Plasmid pBIOS 273

[0079] The basic vector for the homologous recombination is the vectorPBIOS 273. This vector was generated in 2 steps:

[0080] Cloning of the BspDI/XhoI fragment (pAct-Bar-terNos) of thevector pDM 302 (Cao et al., 1992) into the SmaI and BspDI sites of thevector pSB12 (Japan Tobacco). The vector resulting from this cloning iscalled pBIOS 272.

[0081] Deletion of the XhoI site at position 3363 of the vector pBIOS272 by partial digestion with XhoI and the action of DNA Polymerase Ilarge (Klenow) fragment. The vector obtained, which has a unique XhoIsite, is named pBIOS 273.

[0082] Generation of the Intermediate Recombination Vectors Containingthe Asr1 cDNA in the Sense or Antisense Direction

[0083] These constructs were generated from the vector pBIOS 274,derived from the vector pBIOS 273 by cloning the XhoI fragment(ProA9-Barnase-terCaMV) of the vector pWP 128 (Paul et al., 1992) intothe XhoI-restricted vector pBIOS 273.

[0084] The intermediate vectors pBIOS 308 and pBIOS 309 were obtained bycloning the 2970 bp SalI/XhoI fragments of the Vectors pBIOS 306 andpBIOS 307 into the BspDI/XhoI sites of the vector PBIOS 274.

[0085] This cloning thus makes it possible to substitute thepA9-Barnase-terCaMV gene of the vector pBIOS 274 with the pAct-ZmAsr1sense-terNos gene, the resulting vector being called pBIOS 308 (FIG. 2),or with the pAct-ZmAsr1 antisense-terNos gene, the resulting vectorbeing called pBIOS 309 (FIG. 3).

[0086] c) Construction of the Superbinary Transformation Vectors forExpression of the Asr1 Gene and of Its Antisense in Agrobacteriumtumefaciens and in Maize Plants

[0087] Construction of the Superbinary Vectors

[0088] The vectors used for the transformation of maize are derived fromhomologous recombination of the plasmids pBIOS 308 and pBIOS 309 withthe vector pSB1 (EP 672 752). The vector pSB1 contains the virB and virGgenes of the Ti plasmid pTiBo542 present in the Agrobacteriumtumefaciens strain A281 (ATCC 37349), the tetracyclin resistance gene,an origin of replication functional in E. coli and Agrobacterium, and ahomologous region found in the intermediate vectors pBIOS 308 and pBIOS309. The presence of this homologous region in the recipient plasmid(pSB1) and the intermediate plasmids (pBIOS 308 and PBIOS 309) isresponsible for the phenomenon of homologous recombination.

[0089] The intermediate vectors pBIOS 308 and pBIOS 309 are introducedinto the Agrobacterium tumefaciens cells containing the vector pSB1 byelectroporation using the GIBCO BRL CELL PORATOR Voltage Boosteraccording to the method described by Mattanovitch et al. (1989) and theprotocol provided by the supplier (Life Technologies, USA).

[0090] The agrobacteria containing the superbinary vectors are selectedon YT CaCl₂ medium in the presence of rifampicin and spectinomycin at aconcentration of 50 mg/l. The rifampicin resistance gene is carried outby the bacterial chromosome. The spectinomycin resistance, carried bythe plasmids pBIOS 308 and pBIOS 309 (origin of replication in E. coli),may be expressed only after homologous recombination with the vectorpSB1 (origin of replication functional in Agrobacterium and E. coli).

[0091] The superbinary plasmids obtained after recombination are namedpRec 308 (PBIOS 308×pSB1) and pRec 309 (pBIOS 309×pSB1). They possessorigins of replication which are functional both in E. coli and inAgrobacterium tumefaciens, the tetracyclin resistant and thespectinomycin resistant genes, the T-DNA in which are located thecassettes of expression of the Bar and Asr1 (sense or antisense cDNA)genes, and the virB and virG virulence genes of the plasmid pTiBo542.

[0092] Characterization of the Superbinary Vectors pRec 308 and pRec 309

[0093] These superbinary plasmids pRec 308 (Asr1 gene in the sensedirection) and pRec 309 (Asr1 gene in the antisense direction) arecharacterized by enzymatic restriction with SalI. Southern blottinganalysis of the SalI restriction fragments is then carried out with theBar probe and the Asr1 gene probe (this probe corresponds to the 795 bpEcoRI/XhoI fragment of the vector pHHU516, therefore to the completecDNA). The profiles obtained are those expected.

Example 4

[0094] Maize Plant Transformation

[0095] The maize plant transformation is carried out according to theprotocol of Ishida et al. (1996).

[0096] The transformation begins with a co-culture in which the immatureembryos of the maize plants (size ranging from 1 to 1.2 mm) are broughtinto contact, for 5 minutes, with Agrobacterium tumefaciens LBA 4404containing the superbinary vectors pRec 308 or pRec 309. The embryos arethen placed on LSAs medium for 3 days in the dark and at 25° C.

[0097] The following step is that of the first selection of thetransformed calluses: the “embryo-calluses” are transferred onto LSD 5medium containing phosphinotricine at 5 mg/l and cefotaxime at 250 mg/l(elimination of Agrobacterium tumefaciens). This step is carried out 2weeks in the dark and at 25° C.

[0098] The second selection step is carried out by transferring theembryos which have developed on LSD 5 medium onto LSD 10 medium(phosphinotricine at 10 mg/l) in the presence of cefotaxime, for 3 weeksunder the same conditions as in the first selection (25° C., in thedark).

[0099] The third selection step consists in excising the type I calluses(fragments of 1 to 2 mm) and in transferring them into the dark for 3weeks at 25° C. on LSD 10 medium in the presence of cefotaxime.

[0100] The regeneration of the plantlets is then carried out by excisingthe type I calluses which have proliferated and transferring them ontoLSZ medium in the presence of phosphinotricine at 5 mg/l and cefotaxime,for two weeks at 22° C. and under continuous light. The plantlets whichhave regenerated are transferred onto rooting medium (Ishida et al.,1996) for two weeks at 22° C. and under continuous illumination for thedevelopment step.

[0101] The plants obtained are then transferred to the phytotron for thepurpose of acclimatizing them.

Example 5

[0102] Demonstration of Expression of the ZMASR1 Protein in theTransformed Plants

[0103] The proteins of the leaf samples originating from the transformedplants were extracted according to the method of Damerval et al. (1986),and were analyzed by two-dimensional electrophoresis (TDE) according tothe protocol of Riccardi et al. (1998).

[0104] Two-dimensional electrophoresis consists in separatingpolypeptides as a function of their isoelectric point and as a functionof their molecular weight (electrophoresis in the presence of SDS).Before electrophoresis, the proteins are extracted and kept underdenaturing conditions: the quaternary structure is eliminated. Thevarious polypeptides making up the oligomeric proteins migrateindependently during the two electrophoreses. The gels are then stainedwith silver nitrate.

[0105] The two-dimensional gel thus obtained is compared with thatproduced using proteins of nontransformed A188 plant leaves.

[0106] The results obtained from the plants transformed with the codingsequence placed in the “sense” direction show, by simple visualexamination of these two gels, stronger expression of the ASR1 proteinin the transformed plants compared to the A188 control plants.

[0107] The ASR spot observed on the plants transformed with theantisense construct appear to be still detectable, but less strongly,which shows that the “antisense” transformants express the protein lessthan the control plants.

[0108] This protein analysis therefore demonstrates expression of theASR1 protein in the transformed plants, in an amount which is modifiedcompared to the nontransformed plants.

Example 6

[0109] Measurement of the Tolerance to Water Stress of the TransgenicPlants Obtained According to the Invention

[0110] The resistance to water stress of the transformed plantsaccording to the invention, compared with the control plants, can beassessed using various phenotypic, physiological and/or biochemicalanalytical methods, for particular irrigation conditions under normalconditions (conventional culture with watering) and under conditions ofwater stress.

[0111] By way of example, the water conditions in the field may be asfollows:

[0112] normal irrigation in the irrigated part on the basis of 5 mm perday controlled by tensiometers placed 30, 50 and 70 cm deep;

[0113] restrictive irrigation, with no supply of water, if possible,until 10 days after flowering. At this approximate date, it is decidedto provide water when the stress is judged to be too intense; the rhythmof supply should not exceed 3 mm per day.

[0114] The meteorological data and the irrigation conditions arerecorded.

[0115] The grading carried out at the various periods of flowering andof harvesting consist in measuring:

[0116] a) Measurement of the Tolerance to Stress by PhenotypicObservation

[0117] The genetic analyses carried out beforehand suggested a potentialrole for ASR1 in foliar senescence and protandry (time differencebetween male and female flowering) under conditions of drought. Thesecharacteristics are therefore preferentially studied.

[0118] Foliar senescence can be studied with morphological measurementswhich consist in counting the number of dried up leaves and green leavesat 4 dates 15 days apart, from the date of flowering, or at variousstages of development. When very different behaviors are observedconcerning the rolling up and the color of the leaves, an assessment ismade according to a scale of 0 to 5 (from the most tolerant to the leasttolerant to stress).

[0119] Senescence can also be measured by assaying chlorophyll onsamples of foliar disks from the ear leaf at flowering.

[0120] Protandry, or time difference between the dates of flowering ofthe male plants (presence of pollen) and female plants (bristles comingout) is measured as follows: the dates of appearance of the bristles andof the pollen are noted individually for each plant, and the dynamicsare represented on a curve of percentage of plants having flowered as afunction of time.

[0121] Moreover, various assessments were carried out at harvest inorder to evaluate the effect of the tolerance to stress on grainproduction, in particular: the percentage fertilization (ratio of thenumber of grains per ear/number of fertilizable ovules), the number ofrows per ear, the number of grains per row, the water content of thegrains, the weight of 1000 grains and the number of fusarium infectedplants.

[0122] The use of a nondestructive identification test using basta makesit possible to easily distinguish the plants which are resistant tobasta from the sensitive plants, in each transgenic descendance, theresistant plants containing the Asr gene genetically linked to theselectable marker gene, unless there has been a recombination event.This test consists in swabbing the end of a leaf with a solution ofbasta and observing the resulting phenotype, without the vitality of thewhole plant being threatened: the end of the leaf undergoes necrosis anddries out when the plant is sensitive, or remains green if the plant isresistant. This makes it possible to observe, on the measurements oftolerance to stress, whether the ASR transgene positively influences theresponse to stress as a function of expression of the gene in the sensedirection or in the antisense direction.

[0123] b) Evaluation of the Stress Experienced by the Plants

[0124] Samples are taken from the leaves, in order to measure the ABAcontent (method of Quarrie et al., 1988) and, where appropriate, theexpression of the protein by two-dimensional electrophoresis.

[0125] The stress experienced by the plants can be evaluated bymeasuring the basic water potential using a portable pressure chamber onnontransformed plants, under control conditions and under conditions ofwater stress. Measurements of the relative water content of the leavescan also be made.

[0126] Foliar senescence, which permits a decrease in the surface ofevaporation, and re-mobilization of metabolites to the remainder of theplant, was measured as described above, on adult plants after flowering,by counting the number of leaves of which at least 50% of the surface isdry.

[0127] A significant effect of expression of the ASR protein on theproportion of dry leaves is observed. In fact, when related to their owncontrols, the “sense” events show, at a given time, a greater proportionof dry leaves than “antisense” events (FIG. 4). Similarly, underconditions of water stress, the foliar senescence kinetics measurementsshow that the “sense” events become senescent more rapidly than the“nontransformed” and that the “antisense” events become senescent lessrapidly than the “nontransformed” (FIG. 5).

[0128] The term “sense” event is intended to mean an event derived froman initial step of transformation with an expression cassette containingthe ASR sequence placed in the sense direction. The term “antisense”event is intended to mean an event derived from an initial step oftransformation with a cassette containing the ASR sequence placed in theantisense direction.

[0129] The “sense” events, which become senescent more rapidly than the“nontransformed” under conditions of water stress, therefore exhibit aselective advantage of tolerance to water stress, in particular in thecase of a stress of long duration (decrease in the surface ofevaporation and re-mobilization of metabolites to the remainder of theplant).

[0130] The “antisense” events, which become senescent less rapidly thanthe “nontransformed” under conditions of water stress, also exhibit anadvantage, in particular in the case of a water deficit of shortduration. In fact, these plants will have maintained a greater surfaceof evaporation and will therefore benefit more fully from a subsequentsupply of water (rain or irrigation).

[0131] In addition, the measurements of the stress experienced by theplants (ABA content in leaves) revealed a slight but highly significantstress: respectively 616 and 512 ng ABA/g solids in the stressed andcontrol plants, i.e. an increase of approximately 100 ng of ABA per gramof solids in the stressed plants. This response is the same for the“sense” and “antisense” events, and also for the nontransformed plants.The transformation does not therefore appear to have an effect on theaccumulation of ABA in the leaves.

[0132] These results therefore confirm that, in the presence of a slightwater stress and of low ASR expression in the transformed plants,significant differences are already observed.

[0133] Moreover, the effect of the tolerance to water stress on grainproduction was measured relative to the yield of grain, the weight ofone thousand grains and the number of ears per plant. The resultsobtained with a low water stress show grain yield measurementscomparable between transformed (“sense” and “antisense”) plants andnontransformed plants, taken under conditions of stress or under normalconditions.

[0134] The transformation, firstly, and the tolerance to water stress,secondly, do not therefore appear to affect the grain yield of theplants.

[0135] Plant growth measurements were also taken. The lengths of 3leaves above and below the ear were measured on “sense” and “antisense”plants after flowering, in the absence of water stress. A highlysignificant difference was observed for the 3 leaves:

[0136] for the leaves

[0137] F0, antisense=78.41 cm (p<0.01)

[0138] sense=76.11 cm

[0139] F1, antisense=77.96 cm (p<0.01)

[0140] sense=75.76 cm

[0141] F2, antisense=75.04 cm (p<0.01)

[0142] sense=72.94 cm

[0143] Overall, a significant difference of 2 cm in length is thereforeobserved for these 3 leaves, the leaves of the “sense” plants beingsmaller than those of the “antisense” plants. This decrease in leafgrowth observed in the “sense” events therefore correlates with agreater senescence, the two phenomena resulting in a decreased surfaceof evaporation, which allows the plant to more successfully toleratewater stress.

BIBLIOGRAPHY

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[0145] Altschul et al. (1990), J. Mol. Biol. 215: 403-410

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[0178]

1 7 1 777 DNA Zea mays CDS (85)..(504) 1 tgtcgatcca attgtcacttgctctccctc caacaagcta attaaggccg gtcgtcatcc 60 ctcttctagc tcgttttattatcc atg gcg gag gag aag cac cac cac cac 111 Met Ala Glu Glu Lys His HisHis His 1 5 cac ctg ttc cac cat aag aag gac gag gag cag gag gag cag ctcgcc 159 His Leu Phe His His Lys Lys Asp Glu Glu Gln Glu Glu Gln Leu Ala10 15 20 25 ggc ggc ggg tac ggc gag tcc gcc gag tac acg gag gcc acg gtgacg 207 Gly Gly Gly Tyr Gly Glu Ser Ala Glu Tyr Thr Glu Ala Thr Val Thr30 35 40 gag gtg gtg tcc acg ggc gag aac gag tac gac gag tac aag gag gag255 Glu Val Val Ser Thr Gly Glu Asn Glu Tyr Asp Glu Tyr Lys Glu Glu 4550 55 aag cag cat aag cac aag cag cac ctc ggc gag gcc ggc gcc atc gcc303 Lys Gln His Lys His Lys Gln His Leu Gly Glu Ala Gly Ala Ile Ala 6065 70 gcc ggc gcc ttc gca ctc tac gag aag cac gag gca aag aag gac ccg351 Ala Gly Ala Phe Ala Leu Tyr Glu Lys His Glu Ala Lys Lys Asp Pro 7580 85 gag cac gcg cac cgc cac aag atc gag gag gag gtc gcg gcg gcg gcg399 Glu His Ala His Arg His Lys Ile Glu Glu Glu Val Ala Ala Ala Ala 9095 100 105 gcc gtc ggc tcc ggc ggc ttc gcc ttc cac gag cac cac gag aagaag 447 Ala Val Gly Ser Gly Gly Phe Ala Phe His Glu His His Glu Lys Lys110 115 120 aag gac cac aag gac gcc gag gag gcc ggc ggc gag aag aag caccac 495 Lys Asp His Lys Asp Ala Glu Glu Ala Gly Gly Glu Lys Lys His His125 130 135 ttc ttc ggc tgattgatcc ctcccgtatc gtcgtccctc cccgtgtgct 544Phe Phe Gly 140 acgcgtgcgt gtgtgagagt gatatcgagc gcccgccgtg ttgtgcgcgcgtacgtatgt 604 atgcgctcgt gtgatgcacg aataagcgtg gctacgtaat ctatcgtatgtatacgtgtg 664 tgtatgcatg tgcttgtgta tgatcgtggt acgaggaccg aaaaaatgtatgcaactctg 724 atttacttac atgtttagtt gtttacgttt ctccaaaaaa aaaaaaaaaaaaa 777 2 140 PRT Zea mays 2 Met Ala Glu Glu Lys His His His His His LeuPhe His His Lys Lys 1 5 10 15 Asp Glu Glu Gln Glu Glu Gln Leu Ala GlyGly Gly Tyr Gly Glu Ser 20 25 30 Ala Glu Tyr Thr Glu Ala Thr Val Thr GluVal Val Ser Thr Gly Glu 35 40 45 Asn Glu Tyr Asp Glu Tyr Lys Glu Glu LysGln His Lys His Lys Gln 50 55 60 His Leu Gly Glu Ala Gly Ala Ile Ala AlaGly Ala Phe Ala Leu Tyr 65 70 75 80 Glu Lys His Glu Ala Lys Lys Asp ProGlu His Ala His Arg His Lys 85 90 95 Ile Glu Glu Glu Val Ala Ala Ala AlaAla Val Gly Ser Gly Gly Phe 100 105 110 Ala Phe His Glu His His Glu LysLys Lys Asp His Lys Asp Ala Glu 115 120 125 Glu Ala Gly Gly Glu Lys LysHis His Phe Phe Gly 130 135 140 3 892 DNA Zea mays 3 tgtcgatccaattgtcactt gctctccctc caacaagcta attaaggccg gtcgtcatcc 60 ctcttctagctcgttttatt atccatggcg gaggagaagc accaccacca ccacctgttc 120 caccataagaaggacgagga gcaggaggag cagctcgccg gcggcgggta cggcgagtcc 180 gccgagtacacggaggccac ggtgacggag gtggtgtcca cgggcgagaa cgagtacgac 240 gagtacaaggaggagaagca gcataagcac aagcagcacc tcggcgaggc cggcgccatc 300 gccgccggcgccttcgcact cgtacgtagt cctccgatcg atccgatcct ccttgagtag 360 tatatacatacatgaacgcg ataacgaata atatattaat cgaacgaact gaatgatgat 420 cacggatcacctcgtgtgac gtggacatgc acagtacgag aagcacgagg caaagaagga 480 cccggagcacgcgcaccgcc acaagatcga ggaggaggtc gcggcggcgg cggccgtcgg 540 ctccggcggcttcgccttcc acgagcacca cgagaagaag aaggaccaca aggacgccga 600 ggaggccggcggcgagaaga agcaccactt cttcggctga ttgatccctc ccgtatcgtc 660 gtccctccccgtgtgctacg cgtgcgtgtg tgagagtgat atcgagcgcc cgccgtgttg 720 tgcgcgcgtacgtatgtatg cgctcgtgtg atgcacgaat aagcgtggct acgtaatcta 780 tcgtatgtatacgtgtgtgt atgcatgtgc ttgtgtatga tcgtggtacg aggaccgaaa 840 aaatgtatgcaactctgatt tacttacatg tttagttgtt tacgtttctc ca 892 4 890 DNA Zea mays 4tgtcgatcca attgtcactt gctctccctc caacaagcta attaaggccg gtcatccctc 60ttctagctcg tttcattatc catggcggag gagaagcacc accaccacca cctgttccac 120cacaagaagg acgaggagca ggaggagcag ctcgccggcg gcgggtacgg cgagtccgcc 180gagtacacgg aggccacggt gacggaggtg gtgtccacgg gcgagaacga gtacgacgag 240tacaagaagg aggagaagca gcacaagcac aagcagcacc tcggcgaggc cggcgccatc 300gccgccggcg ccttcgcact cgtacgtagt cctccgatcg atccgatcct ccttgagtag 360tatatacata catgaacgcg ataacgaata atatattaat cgaacgaact gaatgatgat 420cacggatcac ctcgtgtgac gtggacatgc acagtacgag aagcacgagg caaagaagga 480cccggagcac gcgcaccgcc acaagatcga ggaggaggtc gcggcggcgg cggccgtcgg 540ctccggcggc ttcgccttcc acgagcacca cgagaagaag aaggaccaca aggacgccga 600ggaggccggc ggcgagaaga agcaccactt cttcggctga ttgatccctc ccgtatcgtc 660gtccctcccc gtgtactacg cgtgcgtgtg tgagagtgat atcgagcgcc cgccgtgttg 720tgcgcgcgta cgtatgtatg cgctcgtgtg atgcacgaat aagcgtggct acgtaatcta 780tcgtatgtat acgtgtgtgt atgcatgtgc ttgtgtatga tcgtggtacg aggaccgaaa 840aaatgtatgc aactctgatt tacttacatg tttagttgtt tacgtttctc 890 5 814 DNA Zeamays modified_base (793) a, t, c, g, other or unknown 5 gctaattaaggccggtcgtc atccctcttc tagctcgttt tattatccat ggcggaggag 60 aagcaccaccaccaccacct gttccaccat aagaaggacg aggagcagga ggagcagctc 120 gccggcggcgggtacggcga gtccgccgag tacacggagg ccacggtgac ggaggtggtg 180 tccacgggcgagaacgagta cgacgagtac aagaaggagg agaagcagca caagcacaag 240 cagcacctcggcgaggccgg cgccatcgcc gccggcgcct tcgcactcgt acgtagtcct 300 ccgatcgatccgatcctcct tgagtagtat atacatacat gaacgcgata acgaataata 360 tattaatcgaacgaactgaa tgatgatcac ggatcacctc gtgtgacgtg gacatgcaca 420 gtacgagaagcacgaggcaa agaaggaccc ggagcacgcg caccgccaca agatcgagga 480 ggaggtcgcggcggcggcgg ccgtcggctc cggcggcttc gccttccacg agcaccacga 540 gaagaagaaggaccacaagg acgccgagga ggccggcggc gagaagaagc accacttctt 600 cggctgattgatcctcccgt atcgtcgtcc ctccccgtgt gctacgcgtg cgtgtgtgag 660 actgatatcgagcgcccgcc gtgttgtgcg cgcgtacgta tgtatgcgct cgtgtgatgc 720 acgaataagcgtggctacgt aatctatcgt atgtatacgt gtgtgtatgc atgtgcttgt 780 gtatgatcgtggnacgagga ccgaaaaaat gtat 814 6 20 DNA Zea mays 6 tgtcgatcca attgtcactt20 7 20 DNA Zea mays 7 tggagaaacg taaacaacta 20

1. A method for obtaining a plant exhibiting a modified amount of ASRprotein, conferring on it better resistance to water stress compared toa nontransformed plant, comprising the steps consisting in: transformingat least one plant cell with a vector containing an expression cassettecomprising a nucleotide sequence encoding an ASR protein or blocking theexpression of an ASR protein; culturing the cell thus transformed so asto generate a plant containing, in its genome, said expression cassette.2. The method as claimed in claim 1, in which said ASR protein isderived from a cereal, such as maize.
 3. The method as claimed in claim2, in which said ASR protein comprises the sequence SEQ ID No
 2. 4. Themethod as claimed in one of claims 1 to 3, in which said nucleotidesequence is inserted in the sense direction.
 5. The method as claimed inone of claims 1 to 3, in which said nucleotide sequence is a sequenceencoding an ASR protein, which is inserted in the antisense direction.6. The method as claimed in one of claims 1 to 5, in which theexpression cassette comprises a promoter for constitutive expression ofthe sequence encoding ASR, for example the actin promoter-intron.
 7. Themethod as claimed in one of claims 1 to 6, in which the expressioncassette contains a sequence selected from SEQ ID No 1, No 3, No 4 andNo
 5. 8. A plant, or a part of a plant, which can be obtained using themethod as claimed in any one of claims 1 to
 7. 9. A plant, or a part ofa plant, as claimed in claim 8, exhibiting an increase in expression ofthe ASR protein compared to a nontransformed plant.
 10. A plant, or apart of a plant, as claimed in claim 8 or 9, characterized in that it isa field crop plant selected from maize, wheat, rapeseed, sunflower andpeas, in particular maize.
 11. A hybrid transgenic plant which can beobtained using a method comprising: a) obtaining two transgenic plantsaccording to the method of any one of claims 1 to 7, and b) crossingthese plants.
 12. The use of a nucleic acid encoding an ASR protein, forobtaining a transgenic plant exhibiting a modified amount of ASRprotein, conferring on it enhanced resistance to water stress comparedto a nontransformed plant.
 13. The use of a nucleic acid encoding an ASRprotein, or a fragment of this nucleic acid, as a probe or primer foramplification, in order to select transformed plants as claimed in oneof claims 8 to 11, exhibiting better resistance to water stress.